Parallel Partial Purification of Cytoplasmic and Mitochondrial Aconitate Hydratases from Rat Liver

Abstract

A rapid method allowing parallel purification of cytoplasmic and mitochondrial aconitate hydratases of rat liver is described. The two enzymes were purified 25 to 35-fold. The isoelectric points and Km values in the production of cis-aconitate with citrate or D-isocitrate as substrates, were measured for both enzymes. Substrate activation at high concentrations of citrate and D-isocitrate has been observed.The enzyme aconitate hydratase has been found both in the soluble cytoplasm and in the mitochondria of different tissues from several animal species [l-71. Guarriero-Bobyleva and Buffa [8] found that, in rat liver cells, only 2201, of the total aconitate hydratase activity is associated with the mitochondria, whereas the rest of the activity is present in the soluble cytoplasm.For a thorough comparison of the properties of the two enzymatic forms, their puri6cation from the same tissue would be desirable. The present paper describes an attempt a t the parallel purification of cytoplasmic and mitochondrial aconitate hydratases from rat liver. The rat liver has been chosen as the source of the enzymes in consideration of previous work both in witro [8,9] and in wiwo [lO,il]. MATERIAL AND METHODSYoung adult Wistar strain albino male and female rats, fed on a standard diet and water ad libitum, were used. The animals were killed by decapitation and bled. The livers were immediately excised, and a 2001, (w/v) homogenate in ice-cold 2 mM Tris-HC1 buffer pH 7.4. containing 0.25 M sucrose and 2 mM citrate, was prepared. The following operations were performed at 0-2 "C, and in the presence of 2 mM citrate in order to protect the enzyme [12]. After centrifugation of total homogenate at 850 x g for lo min, the mitochondria were isolated from the supernatant by centrifugation at 14 500 x g for 10 min and washed twice with 0.25M sucrose. A crude extract of mitochondrial aconitate hydratase in 2 mM Tris-HC1 pH 7.8, containing 2 mM citrate, was obtained by freezing and thawing essentially as describ-~~

Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein.

“…Our attempts to produce c-aconitase in quantities sufficient for various spectroscopic analyses were unsuccessful when we used variations of published procedures (11)(12)(13). A summary of a typical run of the purification method developed is given in Table 1.…”

Iron‐sulphur clusters as genetic regulatory switches: the bifunctional iron regulatory protein‐1

“…The two proteins display ,~31% identity and ~56% similarity along their entire length. A cytoplasmic homologue of mitochondrial aconitase was long known to exist [16], but its role in cell metabolism was not well understood. Both IRP-1 and c-aconitase are known to be localised on human chromosome 9, and the homology between IRP-1 and mt-aconitase, particularly the 100% identity of amino acids involved in the formation of the enzyme's catalytic core, suggested the possibility that IRP-1 and cytoplasmic aconitase might represent two forms of the same protein [14,15].…”

N-t-Butyl Hydroxylamine, a Hydrolysis Product of α-Phenyl-N-t-butyl Nitrone, Is More Potent in Delaying Senescence in Human Lung Fibroblasts

“…In addition N-hydroxylamines prevented the age-dependent decline in aconitase activity in IMR90. Aconitase is an enzyme essential for the Krebs cycle and highly abundant in mitochondria compared with cytosol (29). Its iron-sulfur cluster is known to be damaged by superoxide radical and ONOO Ϫ (25,30,31).…”